Fitzgerald and Schmidt (1912) and Sears (1916, 1917) have demonstrated that bacteria can produce creatinine and the latter

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1 PRECURSORS TO THE FORMATION OF CREATININE BY BACTERIA C. H. FISH AND T. D. BECKWITH Department of Bacteriology, University of California at Los Angeles Received for publication July 29, 1938 Creatinimne is a urinary excretory product from warm-blooded animals but the mode of its formation is not, as yet, completely understood. Folin (1905) determined that its production was independent of intake of food and protein and of non-protein nitrogen. Moreover, its daily output is normally constant. The amount of creatinine excreted per day is proportional to the muscular development of the individual as shown by Tracy and Clark (1914). Since creatinine may be considered to be dehydrated creatine, it has been assumed that creatine is its forerunner, although findings here have been diverse (Folin, 1914 and Lyman and Bundy, 1917). In addition, arginine, choline, glycine-betaine and cystine have been advanced as precursors of creatine but final evidence of the effectiveness of anyone of these is lacking. It is possible that they may be only stimulants. The complexities of the animal body are such that evaluation of variables frequently becomes impossible and therefore it was decided to turn to simple organisms for our experiments, which have been done with certain bacteria. Fitzgerald and Schmidt (1912) and Sears (1916, 1917) have demonstrated that bacteria can produce creatinine and the latter showed that Proteus vulgaris formed this compound from peptone. In the presence of glucose, with the peptone, the amount of creatinine appearing was increased. Because of the relatively simple structure of the bacterial media, control of variables is easier than in the body of an animal. Creatinine may be related theoretically to acetic acid, glycine, 111

2 112 C. H. FISH AND T. D. BECKWITH sarcosine, guanidine, methylguanidine, glycocyamine, arginine, urea or choline and certain of these were examined to determine their availability to act as precursors of creatinine when exposed to the growth activities of bacteria. Procedure. Following our introductory experiments which were devoted to verification of the observations of Sears (1916, 1917), synthetic media were utilized. The common base for these media was Mueller's (1935) salt solution containing NaCl, K2HP04, CaCl2, MgSO4. 7H20 'and FeCl3*6H20 dissolved in distilled water. This solution was sterilized in the Arnold Steamer. The following amino acids have been included: glycine, dl-alanine, dl-glutamic acid, d-arginine HOl, cystine, dlphenylalanine, dl-tyrosine, dl-aspartic acid, dl-leucine, and these compounds were obtained from Amino Acid Manufactures at the University of California at Los Angeles and were stated to contain not more than per cent of impurities. These substances were used in watery solutions and the concentration of hydrogen-ion was adjusted to 7.0. Sterilization was effected by the intermittent method. Media under test were compounded at time of use by addition of 0.25 cc. of Mueller's solution to 10 cc. of solution of the amino acid. Culture volumes usually were 60 cc. The organisms were from stock cultures in the department of the University and had been previously isolated at various times. Determinations of creatinine were made by the method of Jaff6 (1886). A solution of creatinine chloride of concentration of 100 mlg. creatinine in 100 cc. of distilled water was standardized against one of potassium dichromate. In turn, this was diluted to suit subsequent tests. Saturated aqueous solution of picric acid was tested for purity by the method of Folin and Doisy (1917). A representative test was conducted in the following manner: Five cc. of culture solution plus 5 cc. of alkaline picrate stood for five minutes and then were diluted volumetrically to 25 cc. with distilled water. By a colorimeter this solution was compared with 5 cc. of the standard dilution of creatinine to which had been added 5 cc. of alkaline picrate. This, after standing for five minutes, in turn was diluted volumet-

3 PRECURSORS TO CREATININE FORMATION rically to 25 cc. with distilled water. If culture solutions were turbid, they were centrifugalized to prevent interference with readings in the colorimeter. EXPERIMENTAL Experiment 1. Since Sears (1916) stated that Staphylococcus aureus, Proteus vulgaris and Escherichia coli produce creatinine in peptone water and that this creatinine is increased in amount in the presence of glucose, his work was duplicated with results TABLE 1 Production of creatinine by three organisms listed Amounts are expressed as milligrams in 100 cubic centimeters of media E. COWLI. AUREUS P. VULGARIS 3 days 14 days 3 days 14 days 3 days 14 days Peptone water Peptone water + 1 per cent glucose TABLE 2 Production of creatinine by three organisms listed Amounts are expressed as milligrams in 100 cubic centimeters of media. Incubation period was four days 0.1 PER CENT GLYCINE 0.5 PER CENT GLYCINE S. aureus... No growth No growth E.coli P.vulgaris Solution of sodium picrate indicated in table 1. Thus, the work of Sears (1916) has been confirmed and it is indicated that P. vulgaris is more active than E. coli or S. aureus in formation of creatinine. Experiment 2. Since peptone contains a complex of amino acids, the next step was to consider one of the simplest of these, glycine, as a source for formation of creatinine when the three organisms used in experiment 1 were placed in contact with it. For this purpose solutions of glycine of 0.1 and 0.5 per cent concentration were utilized. Results are set forth in table

4 114 C. H. FISH AND T. D. BECKWITH Since sodium picrate, because of the color of its solution, gave an apparent reading of 0.37, it is obvious that a trace only of creatinine was produced in this test. These results indicated that further work was likely to deal with small differences and caused us in certain instances to modify the technique by adding standard creatinine solution to the unknown. The procedure of McCrudden, and Sargent (1916) as indicated below, was employed; Unknown Standard 5 cc. culture solution 5 cc. distilled water 5 cc. standard solution 5 cc. standard solution 5 cc. alkaline picrate 5 cc. alkaline picrate Wait 5 minutes Dilute to 25 cc. volumetrically Read by colorimeter In this experiment, glycine served as the sole source of C and N for the bacteria and of itself was insufficient for formation of creatinine. - Experiment S. Glycine corresponds only to that portion of the molecule of creatinine which contains N-CH2.COOH. Since glycine can serve in such limited degree, it is likely that a precursor corresponding to the HN==C-NH2 portion of the molecule of creatinine might be lacking. Urea might serve this purpose. Therefore various combinations of solutions of glycine, urea, ammonium sulphate and glucose were combined to constitute the series presented in table 3 with Proteus vulgaris to serve as the test organism. Ammonium sulphate was introduced into this series to serve as a control. To include ammonium carbonate would have incorporated an additional source of carbon and that was undesirable. A portion of each of these formulae was kept uninoculated until the conclusion of the experiment and then was tested for possible presence of creatinine. Results were negative in each instance. Since the results obtained by use of glycine, urea and glucose were so exceptional when compared to other units of the series,

5 PRECURSORS TO CREATININE FORMATION 115 the work with this particular combination was repeated. Readings were made at frequent intervals during incubation at 370C. Creatinine appeared in a concentration of 1.6 milligrams per 100 cubic centimeters in twenty-four hours. At five days, it was 6.66 and at fourteen days, 5.0. A mixture of glycine, urea and glucose produced the largest yield of creatinine by Proteus; while all other solutions used permitted formation of little or none of this by-product. Experiment 4. Arginine contains the guanidine group and thus likewise may serve as a precursor to the formation of cre- TABLE 3 Production of creatinine by Proteus vulgaris when in presence of glycine, ammonium sulphate and glucose in various combinations urea Results indicated as milligrams per 100 cubic centimeters MRDIUM 4 DAYS 7DAYS 14 DAYS 0.1 per cent glycine per cent glycine + 1 per cent glucose per cent glycine per cent urea per cent glycine + 1 per cent glucose per cent urea Not tested per cent glycine + 1 per cent glucose per cent ammonium sulphate... Not tested per cent glycine per cent ammonium sulphate per cent glucose per cent urea per cent urea (nogrowth) per cent ammonium sulphate + 1 per cent glucose per cent ammonium sulphate (no growth) atinine. Therefore the series presented in table 3 was set up, with arginine substituted for glycine. With the exception noted, all conditions were identical in the two experiments. The results of these tests are presented in table 4. It may be concluded that arginine is not so effective as is glycine for production of creatinine by Proteus. Moreover an examination of tables 3 and 4 indicates that in the presence of the amino acid and glucose, ammonium sulphate cannot replace urea for formation of creatinine. Experiment 5. Inasmuch as glycine and arginine may serve

6 116 C. H. FISH AND T. D. BECKWITH with urea and glucose as sources for formation of creatinine, it was determined to examine other amino acids individually for like effect. These were incorporated with 0.5 per cent urea and 1 per cent glucose in the synthetic medium described previously and the results are shown in table 5. TABLE 4 Production of creatinine from d-arginine HCl by Proteus Concentrations presented as milligrams per 100 cc. medium MEDIUM 3 DAYS 7 DAYS 14 DAYU 0.1 per cent d arginine HO per cent d arginine H per cent glucose per cent d arginine H per cent urea per cent d arginine HOI per cent ammonium sulphate per cent d arginine H per cent urea + 1 per cent glucose per cent d arginine H per cent glucose per cent ammonium sulphate TABLE 5 Production of creatinine by Proteus from a series of amino acids in presence of glucose Results are expressed as milligrams per 100 cc. of medium AMINO ACID IN MEDIUM 3 DATS 14 DATS 0.1 per cent Glycine per cent dl Alanine per cent dl Glutamic acid per cent Cystine per cent Phenyl alanine per cent dl Leucine per cent dl Tyrosine per cent dl Aspartic acid per cent d Arginine Evidently creatinine can be formed by Proteu8 from all of these amino acids although glycine, cystine, aspartic acid and tyrosine are the most effective of the series. Experiment 6. In the work described thus far, glucose has been the form of carbohydrate utilized. It was desirable to

7 PRECURSORS TO CREATININE FORMATION 117 determine whether another carbohydrate may be substituted for this hexose. A series identical to that given in table 3 except that a pentose, arabinose, replaced glucose was therefore inoculated with Proteus. All other conditions were similar. Final readings with the combination of glycine, urea and arabinose indicated the appearance of 0.22 milligram of creatinine per 100 cubic centimeters rather than 3.0 which were obtained when glucose was present. Subsequent work showed galactose to afford 40 to 65 per cent of the effectiveness of glucose. Maltose upon hydrolysis yields two molecules of glucose but when this TABLE 6 Production of creatinine by bacteria in media described Readings in terms of milligrams per 100 cc. of solution 0.1 PER CENT 0.1 PER CENT D PEPTONE PEPTONE GLYCINE + 1 ARGININE HC1 ORGANISM WATER WATER + 1 PER PER CENT GLU- + 1 PER CENT CENT GLUCOSE COSE PER GLUCOSE CENT UREA PER CENT UREA Proteus Proteus X Alcaligenes Mycobacterium phlei Mycobacterium smegmatis Bacillus subtilis Aerobacter aerogenes Escherichia communis Escherichia communior Escherichia lactici-acidi disaccharide in turn replaced glucose in the media, the yield of creatinine was but 67 per cent of that produced by the monosaccharide. In further work arginine was substituted for glycine and arabinose for glucose. Again it was indicated that the presence of glucose is essential for production of the larger amounts of creatinine. Experiment 7. Three organisms were considered in the first studies, while Proteus alone was used for those designed to determine production of creatinine. It was decided to amplify these findings by inclusion of additional forms. For this purpose, ten bacterial species were selected and with these was incor-

8 118 C. -H. FISH AND T. D. BECKWITH porated Proteus vulgariq to serve as a standard for comparison. Four media were utilized. Final readings of the concentration of creatinine and description of the media are presented in table 6. Incubation extended over a period of fourteen days at 370C. These results indicate that creatinine in considerable concentration may be produced by a variety of bacterial species. With one exception it is demonstrated that more of the by-product is formed in the presence of peptone than with glycine alone and that in many instances, formation of creatinine is intensified by the presence of glucose. Experiment 8. It was demonstrated in table 4 that small amounts of creatinine may be produced from arginine. The presence of urea was repeatedly shown to be necessary if con- TABLE 7 The effect of manganese sulphate upon production of creatinine by Proteus Concentrations noted as milligrams per 100 cc. of medium MEDIUM 3 DATS 14 DAYS 0.1 per cent d Arginine HOl + 1 per cent Glucose per cent d Arginine HOl + 1 per cent glucose + 5 per cent of 0.1 Mol. MnSO per cent Glycine + 1 per cent Glucose per cent urea per cent Glycine + 1 per cent Glucose per cent urea + 5 per cent 0.1 Mol.MnSOa siderable concentrations of this compound are to appear. Arginine contains within its molecular structure two portions: HN2 and a moiety of urea = a guanidine group HN = C\NH NH2 C\NH The question then arises as to whether either of these I is related to the production of creatinine by Proteus. Arginase breaks down arginine with the formation of urea and ornithine. Thus, it destroys the guanidine fraction. Hellerman and Perkins (1935) have demonstrated that salts of manganese and of cobalt

9 PRECURSORS TO CREATININE FORMATION may serve as activators of arginase. It was determined to observe whether the presence of an activator of arginase would stimulate formation of increased amounts of creatinine. Preliminary trials indicated that the cobalt would interfere but that manganese would not interfere with the reading of the reaction. Therefore, the following series presented in table 7 was set up and results as indicated after fourteen days incubation at 370C. were obtained. From these data it is evident that the destruction of the guanidine portion of the molecule of arginine with resultant formation of urea gives rise to the appearance of increased concentration of creatinine when exposed to the enzymatic action of Proteus. Additional evidence regarding the participation of urea in formation of creatinine is adduced. DISCUSSION AND CONCLUSION Creatinine can be produced from peptone by a variety of bacteria but the peptone must be considered as a complex structure involving many factors. Further consideration has proven that creatinine may be formed through bacterial action from various amino acids when glucose is present. In seeking for its precursors, creatinine has been treated as a compound containing two critical portions which are (1) acetic acid and (2) guanidine. It has been shown that glycine, urea and glucose when under the influence of Proteus vulgaris give rise to considerable concentrations of creatinine. Other amino acids than glycine supply either the acetic acid moiety or urea necessary for the reaction. Added evidence regarding the importance of urea is presented by treatment of arginine by an activator of arginase. For production of creatinine by P. vulgaris urea may not be substituted by a salt of ammonium. The hexose glucose is the form of carbohydrate most readily utilized by the organism in the course of creatinine production. The precursors of creatinine as produced by Proteus are acetic acid moiety and urea or glycine and urea. We are indebted to Dr. V. R. Goddasd for her advice in this work. 119

10 120 C. H. FISH AND T. D. BECKWITH REFERENCES FITZGERALD, J. G. AND SCHMIDT, C. L. A Production of creatinine by bacteria. Proc. Soc. Expt. Biol. and Med., 10, FOLIN, 0. K Laws governing the chemical composition of the urine. Am. Journ. Physiol., 13, FOLIN, 0. K On the preparation of creatine, creatinine and standard creatinine solutions. Journ. Biol. Chem. 17, FOLIN, 0. K. AND DoisY, E. A Impure picric acid as a source of error in creatine and creatinine determinations. Journ. Biol. Chem., 28, HELLERMAN, L. AND PERKINS, M. E Activation of enzymes. III. The role of metal ions in the activation of arginase. The hydrolysis of arginine induced by certain metal ions with urease. Journ. Biol. Chem., 112, JAFFA, P Ueber den Niederschlag welchen Pikrinsfiure in normaleor Harn erzengt und fiber eine neue Reaction des Kreatinins. Zeit. physiol. Chem., 10, 391. LYMAN, J. F. AND TRIMBY, J. C The excretion of creatine and creatinine parenterally introduced. Journ. Biol. Chem., 29, 1-5. MCCRUDDEN, F. H. AND SARGENT, C. S The influence of the color from the sodium picrate in the determination of creatinine in blood and urine. Jour. Biol. Chem. 28, MUELLER, J. H Studies on cultural requirements of bacteria. Journ. Bact., 30, SEARS, H. J Studies in the nitrogen metabolism of bacteria. Journ. Inf. Dis., 19, SEARS, H. J Creatinine production by B. coli and B. typhi. Journ. Bact., 2, TRACY, M. AND CLARK, E. E The excretion of creatinine by normal women. Journ. Biol. Chem. 19,